Association Between Single Nucleotide Polymorphisms and Viral Load in Congenital Cytomegalovirus Infection

Abstract Background There are limited data on factors that determine viral load (VL) in congenital cytomegalovirus (cCMV) infection. Single nucleotide polymorphisms (SNPs) might influence individual host response to infection. This study aimed to investigate the association between SNPs in genes encoding cytokines or cytokine receptors and VL in newborns with cCMV. Material and methods Eight polymorphisms (IL1B rs16944, IL12B rs3212227, IL28B rs12979860, CCL2 rs1024611, DC-SIGN rs735240, TLR2 rs5743708, TLR4 rs4986791 and TLR9 rs352140) were analyzed in study population of 233 newborns, including 92 cCMV-infected newborns (73 symptomatic and 19 asymptomatic) by TaqMan SNP Predesigned Genotyping Assays. The association analysis was performed using SNPStats software and STATISTICA10. Results The association between IL12B polymorphism and viruria was observed (p = 0.029). In multiple comparison tests, heterozygous T/G genotype of IL12B was associated with higher viruria than T/T genotype (p = 0.041) in cCMV-infected newborns. In allele analysis, T allele of IL12B was associated with higher viremia (p = 0.037) in symptomatic newborns. We observed higher VL in symptomatic newborns in comparison to asymptomatic (median viremia: 1.7 × 104 copies/mL vs. 2.0 × 103 copies/mL (p = 0.002), median viruria: 1.0 × 107 copies/mL versus 6.9 × 105 copies/mL (p = 0.001), respectively). Conclusions IL12B rs3212227 was associated with VL in cCMV. Symptomatic newborns had significantly higher viremia and viruria. The role of SNPs in pathogenesis of cCMV warrants further investigations.


Introduction
Human cytomegalovirus (HCMV) causes the most common mother-to-a-child intrauterine infection. Congenital cytomegalovirus (cCMV) infection concerns 1% of newborns, which results in about 40,000 infected newborns in the United States each year. 1 cCMV is diagnosed when HCMV is detected in newborn urine until 21st day of life by qualitative real-time polymerase chain reaction (PCR). 2 Viral load (VL) in whole blood (viremia) and urine (viruria) is usually measured at diagnosis and during antiviral treatment in cCMV infection. Infants with cCMV infection have significantly higher viruria than infants with postnatal CMV (pCMV) infection, acquired through the birth canal or breast milk. 2,3 Moreover, women who transmit HCMV to neonate during prenatal period have higher HCMV DNA levels in urine, blood and amniotic fluid. 4, 5 It has been suggested that higher VL could be a marker of the increased risk of significant neurological sequelae in cCMV infection. 6 However, the real predictive value of VL for short-and long-term outcomes in cCMV infection is still unclear. There are limited data on factors that affect the level of viremia and viruria in cCMV-infected newborn infants. Monocytes, cytokines and pattern recognition receptors (PRRs) seem to be the first line of host immune defense against HCMV. 7 Single nucleotide polymorphisms (SNPs) in genes encoding cytokines and cytokine receptors might

Qualitative and quantitative detection of HCMV DNA
The presence of HCMV DNA was evaluated primarily in urine and blood by qualitative real-time PCR as described previously. 11 Viremia and viruria were determined in HCMV DNA positive samples by using the Gene Proof Cytomegalovirus PCR Kit (Gene Proof, Brno, Czech Republic), which amplifies a region of gene encoding the 4 IE antigen. Amplification was performed according to the manufacturer's instructions using a LightCycler 480 II Instrument (Roche, Indianapolis, IN). The results were expressed as copies/mL.

Candidate genes selection
SNPs in eight candidate genes were selected a priori based on previously reported associations in HCMV infection. 8-14 TLR-2 (Toll-like receptor), TLR-4, TLR-9 and DC-SIGN (dendritic cell-specific ICAM-grabbing non-integrin) were chosen for their role in HCMV recognition and triggering inflammatory cytokines. 10, 16-20 IL-1B, IL-12B and IL-28B were chosen for their pro-inflammatory and immunomodulatory roles in the context of HCMV infection. 11, 13, 21, 22 Finally, CCL2 (C-C motif chemokine ligand 2), a monocyte chemotactic protein-1 (MCP-1), was chosen due to its role in immune cell differentiation and their migration during inflammation in HCMV infection. 23

Determination of SNP genotypes
SNPs genotyping was performed using genomic DNA extracted from whole blood collected for qualitative real-time PCR reaction. TaqMan SNP Predesigned Genotyping Assays (Applied Biosystems, Inc., Foster City, CA, USA) were applied to a panel of eight polymorphisms (IL1B rs16944, IL12B rs3212227, IL28B rs12979860, CCL2 rs1024611, DC-SIGN rs735240, TLR2 rs5743708, TLR4 rs4986791 and TLR9 rs352140). The allelic discrimination was performed on the 7500 Real-time PCR System (Applied Biosystems) according to the manufacturer's instructions. A blinded duplicated genotyping of 10 random study samples demonstrated 100% concordance.

Statistical analysis
The normality of the distribution of analyzed data was assessed by Shapiro-Wilk test. Thus, data were presented as median and interquartile ranges (IQR). Non-parametric tests (Mann-Whitney and Kruskal-Wallis ANOVA) were used due to the non-normal distribution of analyzed variables. The categorical variables were presented as numbers and percentages, and Pearson's Chi-squared test was used for comparisons. The association analysis was performed using SNP Stats software 24 and STATISTICA10. The analysis of the association between eight selected SNPs and VL was calculated separately for cCMV-infected, symptomatic and asymptomatic newborn infants. All SNPs (cCMV-infected and healthy control groups) were analyzed for Hardy-Weinberg equilibrium (HWE) by using the Chi-square test. p ≤ 0.05 was considered significant.

Distribution of SNP genotypes
Genotype distribution of examined SNPs was obtained in HWE in the entire study population (comparison between healthy control group and cCMV-infected infants as well as symptomatic and asymptomatic infants). We found no significant differences in genotype distribution of assessed SNPs between symptomatic and asymptomatic newborn infants (Table S1 in Supplementary Materials). Results of genotype distribution have been previously presented for cCMV-infected and healthy control group and also at least partially for symptomatic and asymptomatic cases 15

Frequencies of SNP alleles
Allele frequencies of examined SNPs were obtained in HWE in the entire study population (comparison between healthy control group and cCMV-infected infants as well as symptomatic and asymptomatic infants) (Tables S2-S3 in Supplementary Materials). No significant differences were found in allele frequencies of assessed SNPs in both above configurations.

Association between SNP alleles and VL in blood and urine
We analyzed the association between alleles of eight examined SNPs and VL in blood and urine in 92 neonates with cCMV infection including 73 symptomatic and 19 asymptomatic infants. We observed the association between T allele of IL12B rs3212227 polymorphism and higher viremia in symptomatic infants only (p = 0.037;

Discussion
This prospective study investigated associations between eight SNPs in genes encoding cytokines and cytokine receptors (IL1B rs16944, IL12B rs3212227, IL28B rs12979860, CCL2 rs1024611, DC-SIGN rs735240, TLR2 rs5743708, TLR4 rs4986791 and TLR9 rs352140) and VL in blood and urine in newborn infants with cCMV infection. We found the novel associations between IL12B polymorphism and VL in neonates with cCMV infection. We observed that heterozygous T/G genotype of IL12B rs3212227 polymorphism was associated with higher viruria than T/T genotype in cCMV-infected neonates. The strength of these results was not supported by alleles analysis. Neither T nor G allele of IL12B rs3212227 promoted higher viruria separately. We assume that probably only heterozygous T/G status (both alleles altogether) uphold increased HCMV DNA concentration in urine. Interestingly, T allele of IL12B rs3212227 was associated with higher viremia in symptomatic neonates. However, neither T/T nor T/G genotype was connected with higher VL in blood as could be expected. Popescu et al. proved that impaired CMV-specific T helper (Th) cellular immunity is highly dependent on IL-12. 25 IL-12 is an inflammatory cytokine that initiates T-cell differentiation towards Th1 and inhibits Th2 lymphocyte activity. 26 So far, SNPs of IL-12 were found to be associated with other viral infections, such as hepatitis B virus (HBV), hepatitis C virus (HCV) and also with HCMV infection among adult patients. 27-29 Seegers et al. link IL12 rs3212227 SNP polymorphism with increased IL-12 production. 30 We only might speculate that newborn infants carrying heterozygous T/G genotype of IL12B rs3212227 produce higher amount of IL-12. Presumably, IL-12 could promote higher urinary HCMV excretion to eliminate the virus. Our study might support a hypothesis on the protective role of IL12 polymorphism in a host defense against HCMV infection. However, we could not explain contradictory data of T allele and T-linked genotypes in relation to viremia. It should be underlined that the number of newborns in each subgroup was relatively small, despite the fact, it was one of the largest population of cCMV-infected neonates that was genetically studied so far. Certainly, further investigations are warranted to confirm the association between SNP of IL12B rs3212227 and higher viruria and verify the role of T allele in HCMV DNA concentration in blood.
There is still no consensus whether viruria is a result of kidney virus filtering during viremia or HCMV origins from infected renal cells. HCMV kidney tropism was widely documented, mostly among renal allograft recipients. 31 In relation to cCMV infection, the highest amount of HCMV was observed in pancreas, liver, kidney and brain of the fetus, which suggested a disseminated HCMV infection. 32 On the other hand, cCMV-infected infants tend to excrete virus in the urine for even 3-5 years, 33 even if HCMV is not detected in the blood. Our results may support previous findings showing that viruria might be less likely to be secondary to HCMV DNA concentration in blood than to renal cells infection. Symptomatic infants with cCMV infection excrete higher amount of HCMV DNA in urine than asymptomatic infants. 6 We also observed significantly higher viruria and viremia in symptomatic infants. It should be underlined that higher VL can be linked to an increased risk of unfavourable sequelae of cCMV infection. 34 HCMV viremia was also noted in heterozygous genotype of TLR7 rs5741880. 39 However, above-mentioned reports included both children with cCMV and pCMV infection. As far as cCMV infection was concerned, no associations were found between TLR2 2258 G>A SNP (rs5743708) and HCMV VL in neonates, but G/A variants in TLR2 2258 and TLR9 2848 C>T (rs352140) SNPs were correlated with higher VLs in fetal amniotic fluid and maternal urine samples. 19 In our study, none of three examined TLR SNPs (TLR2 rs5743708, TLR4 rs4986791, TLR9 rs352140) were associated with higher viremia or viruria. We also found no association between CCL2 and DC-SIGN SNPs and VL in blood and urine.
We compared allele frequencies of our study population to the reference population from the Reference SNP Report. 40 As we expected, our study population allele frequencies were very similar to the frequencies in the reference European population (the difference level did not exceed 5 percentage points) ( Table S1 in Supplementary Materials). Some limitations of our study should be acknowledged. We are aware that there are some confounding factors Table 3. Association between alleles of examined polymorphisms (SNPs) and viremia and viruria in symptomatic newborn infants with cCMV infection. which may affect the results, but the current study was not designed to address these issues. First, the genetic background and the serological status of the mother are relevant for the risk of primary infection. Undoubtedly, the type of maternal infection (primary or non-primary) and the time of fetal infection may impact the genetic study results. Moreover, the diversity of HCMV strains may affect the results of the study. Although the current study population is larger than in our previous report, 11 the sample size might be still too small for genetic associations. In addition, no correction for multiple testing was applied, and so type I error cannot be completely ruled out.

Symptomatic newborn infants with cCMV infection (N
The important strength of our study is its prospective design. Furthermore, we included homogeneous population consisted of Caucasian central European newborn infants with cCMV infection, what let us eliminate potential race dependent genetic factors. We focused on congenital infection only because we thought that different genetic mechanisms might determine congenital and postnatal HCMV infection. What is more, five of eight selected SNPs were analyzed for the first time in the context of cCMV infection. 15 To the best of our knowledge, this is the first and the largest study that analyzed the association between the eight SNPs and VL, and the first study that suggests the novel association between IL-12B polymorphism and VL in cCMV infection. However, future studies are necessary to replicate these findings in the larger study population.
To summarise, we found the novel associations between IL-12B polymorphism and VL in cCMV-infected newborns. Our results confirmed that symptomatic newborn infants had higher viremia and viruria than asymptomatic newborn infants.
Indisputably, the role of SNPs in the pathogenesis of cCMV requires further investigations, which may change prognosis for newborn infants with cCMV in the future.

Key points
SNPs in genes encoding cytokines and cytokine receptors might determine the level of VL in cCMV infection. Association between eight SNPs of IL1B, IL12B, IL28B, CCL2, DC-SIGN, TLR2 and TLR4, TLR9 and VL in cCMV infection was studied. T/G genotype of IL12B rs3212227 was associated with higher viruria than T/T genotype in cCMV infection. T allele of IL12B rs3212227 was associated with higher viremia in comparison to G allele in symptomatic cCMV infection. Symptomatic newborns had significantly higher viremia and viruria.
No other associations between SNPs and VL were reported in newborns with cCMV-infection. SNPs analysis might be suitable for better identification and diagnosis of newborns (or even fetuses) with cCMV infection.

Conflict of Interest/Competing Interests
The authors declare that they have no conflict of interest.